Duchenne Muscular Dystrophy (DMD) is associated with mutations in the dystrophin gene, leading to progressive wasting of striated muscle and early death. Skeletal muscles in DMD patients undergo cycles of myofiber necrosis and regeneration, but over time there is a loss of myofibers and the degenerating myofibers are replaced with fibrotic tissue. Treating DMD requires means for stabilizing the myofiber sarcolemma and for combating fibrosis. Although both fronts have been intensely studied, effective treatments are yet to be developed. The proposed study will analyze the potential of klotho (also known as alpha-klotho) to reduce fibrosis in the dystrophin-null mdx mouse, a murine model of muscular dystrophy. Klotho functions as both a transmembrane protein and a secreted humoral factor. The secreted klotho has been shown to directly inhibit the pro-fibrotic TGF-?1 and Wnt pathways in renal tissue. While the main site of klotho expression is the kidney, it is also expressed in other tissues. Should the anti-fibrotic effect of klotho extend to he muscle tissues, the klotho protein could become an intriguing candidate for reducing muscle fibrosis in dystrophin-deficiency where a role for TGF-?1 and Wnt in muscle fibrosis has been established. One main aim of the proposed project is to gain insight into the potential of klotho transgenic (Kt) overexpression to alleviate muscle fibrosis in mdx mice. In addition to histological assays of fibrosis, calcification and myofiber pathology, the study will examine CD45+ cell level as a measure of inflammation and myogenic (satellite cell) activity that appears to be impaired by fibrosis. A second aim is to analyze the effect of the Kt transgene on whole body performance by examining voluntary wheel running, which declines in the mdx mouse. The mdx4cv strain that develops less revertant myofibers than the standard mdx mouse line will be used. Emphasis will be given to mdx and Kt/mdx mouse groups of young to old ages to gain a comprehensive insight as the mdx muscle pathology intensifies with age. Both the mouse lines and the expertise for the proposed studies are already available, permitting effective progress. It is recognized that while the mdx mouse has been used extensively as a laboratory model of DMD, the mdx pathology is less severe than that of DMD patients. Still, studies with mdx mice have generated important directive data for subsequent studies with dystrophic dogs and for pilot clinical trials in humans. The additional inclusion of wildtype (wt) and Kt/wt mouse groups in the proposed study will not only permit comparison with the parental lines, but also allow gaining insights into the effect of the klotho transgene in the wildtype context. The outcome of this exploratory study may identify a new clinical direction for combating DMD and other muscular dystrophies as well as improving muscle quality in aging. The R21 mechanism that is intended to encourage exploratory high-risk high-reward novel studies is the optimal platform for this application.